Mycoscience VOL.62 (2021) 1-9

Review Systematics, ecology, and application of : Recent progress and future perspectives for research with special emphasis on activities within Japan Tsuyoshi Hosoya National Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba-shi, Ibaraki, 305-0005, Japan

ABSTRACT Helotiales is one of the most diverse groups of apothecial ascomycetes, including 3000–4000 taxa. Recent progress in the systematics, ecology, and their applications through research is herein reviewed based on the experiences of the author with a special emphasis on activities in Japan. In the past 30 y, more than 50 helotialean taxa have been added to the mycobiota of Japan, including new taxa. With the advent of molecular phylogeny, some families have been revisited, such as members with stroma ( and Rutstroemiace- ae) or hairs ( and Lachnaceae). Although the monophyly of Helotiales has not yet been demonstrated, our understand- ing of its phylogeny has greatly advanced. The unexpected ecological nature represented by endophytism has been revealed through barcoding and other molecular techniques. The research history of ash dieback is also reviewed, and the endophytism/saprophytism of the pathogen on its original host is discussed. Drug discoveries within Helotiales are reviewed, and successful examples are presented. As future perspectives, both the cumulation of occurrence and sequence data of Helotiales is greatly encouraged to elucidate this important group of fungi.

Keywords: discovery research, Hyaloscyphaceae, Lachnaceae, ,

Article history: Received 5 March 2020, Revised 12 May 2020, Accepted 13 May 2020, Available online 20 January 2021.

1. Introduction high level of diversity, which may be reflected in their met- abolic diversity, but relatively underexplored not only taxonomical- Among the apothecial ascomycetes with inoperculate asci, Hel- ly but also in applied science. 4) They are also thought to be ecolog- otiales is one of the most diverse groups. It is known to include ically diverse, suggesting diverse metabolites. In short, Helotiales some 3000–4000 taxa (Kirk, Cannon, Minter, & Stalpers, 2008; are underutilized but easily accessible fungi (Hosoya, 1998). Baral, 2016). Most members of the Helotiales have minute apothe- I have been working on this attractive group of fungi for more cia, usually less than 2 mm diam. They may be sessile or stipitate, than 30 y, first as a researcher in the private sector in search of nov- dark to bright colored, and superficial or erumpent through the el biological resources for metabolites, and then in a more pure sci- plant host. The overall shape of the apothecia is cupulate-discoid, ence-directed position. Through my career, I have made many new turbinate funnel-shaped, or clavate (Korf, 1973). Most are known to discoveries and contributions to the field of systematics and discov- be saprophytic, living on fallen leaves and decaying wood, but some ery research. In the present article, I review recent advances and are parasitic to pathogenic or symbiotic with other organisms. future perspectives for research on this attractive group of fungi. Many members are relatively easy to isolate, but the inducing the formation of apothecia in vitro is not easy (Müller & Loeffler, 1976). 2. Systematics Though not frequent, some members produce an asexual state, but many remain unassociated with this state. Despite these biological 2.1. Infrastructure for systematics diversities, taxonomy and ecological studies are insufficient. From the viewpoint of applied science, in particular discovery Collecting specimens is a fundamental of systematics. In the “in research, Helotiales are an attractive biological resource for the fol- situ” activity in the National Museum of Nature and Science, the lowing reasons. 1) They are in different ecological groups than soil following process is usually applied. Once the specimens are col- fungi. 2) Most are easily accessible and culturable. 3) They have a lected, single spore isolation using Skerman’s manipulator (Sker- man, 1968) or multisporous isolation from discharged is * Corresponding author. attempted, then the specimens are heat dried or air dried and E-mail address: [email protected] (T. Hosoya) stored as voucher specimens in the fungarium. DNA is extracted

This is an open-access paper distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivative 4.0 international license (CC BY-NC-ND 4.0: https://creativecommons.org/licenses/by-nc-nd/4.0/).

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Ex situ In situ

Other collections Field （Personal, Abandoned） Collecting Acception Materials Processing (format, databasing) Isolation, Cultivation

DNA / tissue samples Specimens Isolates

Fungarium External Culture Collection Deep freezers NITE-NBRC, JCM, etc.

Figure 1. Diagrammatic representation showing the research process and management of voucher specimens.

from the isolates and stored in a freezer as a sample for future use. 1996), and it is now known as a separate group (Orbiliomycetes) When isolation is not successful, part of the apothecia is kept in a from Helotiales and is recognized as a primitive member of the buffer [20% DMSO, 100 mM Tris-HCl (pH8.0), 250 mM EDTA, 100 apothecial fungi (Eriksson, Baral, Currah, Hansen, & Kurtzman,

mM Na2SO3, NaCl to saturation; Hosaka & Castellano, 2008] for 2003; James et al., 2006). Studies are largely limited to Dermateace- DNA extraction when necessary. If asexual reproduction is recog- ae, , and . In my previous studies, I nized in culture, the culture of isolates may also be dried and regis- worked with Hyaloscyphaceae and Sclerotiniaceae sensu lato, tered as specimens. Because our facility is not suitable for the which are reviewed below. maintenance of cultures, we have a close collaboration with cul- ture collection facilities that have the capacity to store intriguing 2.3. Taxonomy of Hyaloscyphaceae isolates from the specimens (Fig. 1). Given the above process, four major collections (specimens, Hyaloscyphaceae is a large family (74 genera + 61 synonyms, isolates, extracted DNA and tissue samples) were obtained and 933 species., Kirk et al., 2008), composed of fungi with minute to managed in-house (http://db.kahaku.go.jp/webmuseum_en/; da- small apothecia with various hairs. The generic taxonomy of these tabase opened for specimens only) or ex-house (e.g., NBRC, https:// fungi was based on a combination of characters, including that of www.nite.go.jp/nbrc/catalogue/NBRCDispSearchServlet? hairs, ectal excipular structure, and paraphyses. Korf (1973) subdi- lang=en; GBIF, Global Biodiversity Information Facility, https:// vided the family into two subfamilies: Trichoscyphelloideae, includ- www.gbif.org/) databases. These collections may be linked with ing a single , , and subfamily Hyaloscyphoideae. sequence data on publicly accessible databases (INSDC, http:// The latter was subdivided into five tribes (Arachnopezizeae, Hyalos- www.insdc.org/). All of these sites were visited on Feb 25, 2020. cypheae, Lachneae, Trichodisceae, and Trichopezizelleae). Most The occurrence data based on the specimens were also provided to members of Hyaloscyphaceae belong to Arachnopezizeae, Hyaloscy- GBIF for global use. Our systematic studies were based on these pheae, Lachneae, and Trichopezizelleae. A number of genera with infrastructures. similar morphology have been identified in , one of the major genera in Lachneae, and even more genera have been pro- 2.2. Advances in the systematics of Helotiales and their stud- posed (e.g., Baral & Kiriegelsteiner, 1985). However, because mor- ies in Japan phological characters often show convergence, revision of generic taxonomy based on phylogeny is warranted. Korf (1973) published a key to the genera of Helotiales with tax- Otani (1989) summarized the known occurrence in Japan and onomic comments. This monumental publication covered a wide listed 41 taxa. However, we expected more undocumented mem- range of taxonomic concepts for apothecial fungi and has been bers. In a series of studies, we contributed to the enumeration of used since the establishment of Helotiales by Nannfeldt (1932). In undocumented taxa, including new species (Hosoya & Otani, his taxonomy, Helotiales were divided into eight families (Ascocor- 1997a, 1997b; Hosoya & Harada, 1999; Ono & Hosoya 2001; Tanaka ticiaceae, , Geoglossaceae, Hemiphacidiaceae, Hyalos- & Hosoya, 2001; Tochihara & Hosoya, 2019). cyphaceae, Leotiaceae, Orbiliaceae, and Sclerotiniaceae), whereas a Cantrell and Hanlin (1997) first conducted a phylogenetic anal- more recent treatment (Baral, 2016) recognized greater taxonomic ysis to assess the taxonomy of Hyaloscyphaceae using a single gene, diversity in 25 families (Table 1). Reviewing recent taxonomic stud- ITS-5.8S. In their analysis, they clarified most of the genera in ies in Japan based on the classification by Korf (1973), Ascocortici- Lachneae, which formed a monophyletic group, but the phylogeny aceae had not yet been documented. Geoglossaceae has been almost of higher clades was less supported. Hosoya et al. (2010c) conduct- completely unstudied since Imai (1934) and is now treated as an ed a multi-gene analysis using the combined data of ITS-5.8S, the independent class apart from Helotiales (Schoch et al., 2009). Orbil- D1-D2 region of LSU, and RPB2, to assess the taxonomic delimita- iaceae has been almost unstudied with a few exceptions (Nagao, tion of the genera in Lachneae. Phylogenetic support of most gen-

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Table 1. Comparisons of recent taxonomy at the family level (Baral, 2016) with the previous concept (Korf, 1973).

Baral (2016) Cladea Korf (1973)b Cenangiaceae Rehm A Hemiphacidiaceae Korf Rutstroemiaceae Holst-Jensen, L. M. Kohn & T. Schumacher A Sclerotiniaceae Sclerotiniaceae Whetzel A Sclerotiniaceae Ascocorticiaceae J. Schröt. Au Ascocorticiaceae Ascodichaenaceae D. Hawksw. & Sherwood Au N/A Chaetomellaceae Johnst. & Rossman Au Hyalocyphaceae Chlorociboriaceae Baral & P. R. Johnst. Au Rehm Cordieritidaceae (Sacc.) Sacc. Au Helotiaceae Dermateaceae Fr. Au Dermateaceae Godroniaceae Baral Au Helotiaceae Mitrulaceae Rchb. Au Geoglossaceae Corda [Stamnaria Fuckel lineage] Au Leotiaceae Corda Gelatinodiscaceae S. E. Carp. B N/A (Helotiaceae) Helotiaceae Rehm s. l. B Helotiaceae Roesleriaceae Y. J. Yao & Spooner B N/A [- lineage] B Leotiaceae Loramycetaceae Debbus ex Digby & Goos Cm N/A Mollisiaceae Rehm Cm Dermateaceae Locq. ex Korf Cm (Ostropales Nannf.) [ Schulzer lineage] Cm Leotiaceae Calloriaceae Marchand Cp Dermateaceae Drepanopezizaceae Bat. & H. Maia Cp Dermateaceae Heterosphaeriaceae Rehm Cp Dermateaceae Ploettnerulaceae Kirschst. Cp Dermateaceae [Hysteropezizella Höhn. lineage] Cp Dermateaceae Arachnopezizaceae Hosoya, J. G. Han & Baral D Hyalocyphaceae Hyaloscyphaceae Nannf. D Hyalocyphaceae Lachnaceae Raitiv. D Hyalocyphaceae Pezizellaceae Velen. D N/A (Hyalocyphaceae) [ L. Ludw., P. R. Johnst. & Steel lineage] D N/A Correspondence between the two are based on the major genera included in the families, sorted on clade, families in Baral (2016) with generic lineage at the end, and Korf (1973). a Clades given by Baral (2016) who disposed each family in A–D clades. Au indicates an uncertain position in clade A. Cm represents Mollisiaceae sublineage in clade C, and Cp represents Ploettnerulaceae sublineage in clade C. b N/A is to indicate that the given type genus in the family was not dealt in Korf (1973), but suggested to be included in the families in the parenthesis based on the synonymy. was treated as a genus in the order Ostropales, and later it was disposed to newly proposed family (Vibrisseaceae in Korf, 1990).

era was obtained, and genera separated from Lachnum were justi- 2.4. Taxonomy of and its allies in Sclerotiniaceae fied. Raitviir (2004) proposed raising the tribe Lachneae to family sensu lato Lachnaceae based on suggested strong monophyly. However, whether the family should include members of Trichopezizelleae is The family Sclerotiniaceae was proposed by Whetzel (1945) for still under debate (Hosoya et al., 2010b), and Ekanayaka et al. members of Helotiales that produce stroma, which included two (2019) included it in their new family Solenopezizaceae. types, sclerotial stroma (sclerotia) forming “a more or less charac- In the continuation of these studies, Tochihara and Hosoya teristic form and a strictly hyphal structure under the natural con- (2019) added three new species to the genus , previ- ditions of its development” and substratal stroma of “diffuse or in- ously known to consist of a small number of species. Studies to definite form,” often recognized as a thin black layer on the surface clarify the generic limitations of these groups are still being con- of the substrate or lines in a vertical section. Both were supposed to ducted, but more data are required to clarify the generic limits to be an overwintering structure to survive low temperatures. Mem- assess the type genus Lachnum. bers of these fungi have been primarily studied in Japan because of On the other hand, the phylogenetic analysis of Hyaloscypheae agricultural interests (e.g., Harada, 1977; Willetts & Harada, 1984). revealed great phylogenetic diversity (Han, Hosoya, Sung, & Shin, Later, Holst-Jensen et al. (1997) illustrated the phylogenetic dif- 2014a). Although they used combined data of ITS-5.8S, the D1-D2 ferences of members with substratal stroma from members with region of LSU, RPB2, and mtSSU, and obtained deeper clades to sclerotial stroma and proposed the family Rutstroemiaceae for support the taxonomy of genera, clades at higher levels were not members with substratal stroma. strongly supported, suggesting poly/paraphyly of the tribe. They The genus Lambertella, first known in Sclerotiniaceae and later hesitated to propose any taxonomic revision for the Hyaloscypheae transferred to Rutstroemiaceae, is chiefly characterized by sub- because it was suggested that the tribe was divided into too many stratal stroma and ascospores that turn brown when mature (Whet- small groups of genera. zel, 1943). Following Whetzel (1943), Dumont (1971) published a Han et al. (2014a) also showed that Arachnopezizeae was no monograph of Lambertella in which 29 species were included, but longer acceptable as a monophyletic group, and proposed Arachno- more potential members were expected because browning of the stat. nov., excluding the heterogeneous elements and ascospores sometimes occurred only after discharge and were over- clarified their phylogenetic differences from the rest of Hyaloscy- looked in the dried specimens. In fact, we made transfers from phaceae. other genera to Lambertella (Hosoya & Otani, 1997c) or justified previous transfers (Hosoya, Otani, & Furuya, 1993) based on the

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observation of fresh specimens. However, browning of the spores more informative data are required to determine the entire taxo- may be a convergent feature and molecular phylogenetic assess- nomic structure of Helotiales, whereas ambitious informal propos- ment is required. als to separate some members in other orders (such as “- Zhao et al. (2016) conducted a molecular phylogenetic analysis les”) have been made (Ekanayaka et al., 2019). of Lambertella, incorporating other members of Rutstroemiaceae, Sclerotiniaceae, and Helotiaceae, based on LSU and RPB2, and 3. Ecology demonstrated the following facts. 1) Sclerotiniaceae is monophylet- ic but is placed within the clade of Rutstroemiaceae. 2) Stromata 3.1. Expanding realm for function of Helotiales in ecosystems have multiple origins, occurring both in Rutstroemiaceae and Helo- tiaceae. 3) Lambertella is a polyphyletic genus that requires taxo- Although some members have been known as plant pathogens nomic revision. Based on the common features observed in the (e.g., Sclerotinia spp.) or symbiotic partners of plants (e.g., Rhizos- strongly supported clade including the type species Lambertella cyphus ericae (D.J. Read) W.Y. Zhuang & Korf), most of the mem- corni-maris Höhn., they redefined the genus Lambertella with ectal bers of Helotiales were considered weak saprophytes in the past excipulum composed of brown, rectangular cells, and ascospores (Alexopoulos, Mims, & Blackwell, 1996). The advent of molecular becoming brown within the asci before discharge (Zhao, Hosaka, & techniques in ecological studies made meta-genomic analysis pos- Hosoya, 2016), in addition to the substratal stroma. Their exclusion sible (e.g., Toju et al., 2013b). As a result, more environmental DNA resulted in several necessary transfers of current members of Lam- data have accumulated, and a wider range of occurrence of Helotia- bertella to appropriate genera, including new genera that needed to les has been suggested in the environment. In recent years, two be proposed, but these have been left untreated to avoid further topics have been remarkable: their function as endophytes and as a confusion under the present unstable taxonomy. Their findings virulent invasive pathogen, fraxineus, causing ash also provided a new concept regarding sclerotia as a special type of dieback. substratal stroma and the merging of Rutstroemiaceae into Sclero- tiniaceae s. l. (Johnston et al., 2019). 3.2. Root endophytes

2.5. Advances in helotialean mycobiota exploitation in Japan In contrast to mycorrhizae, which have a long research history (e.g., Peterson, Massicotte, & Melville, 2004), endophytes have a As already described, exploration and enumeration of mycobio- relatively shorter history in research (e.g., Stone, Polishook, & ta are among the bases of our systematic studies. Otani (1989) White, 2004). Regarding Helotiales (Wang et al., 2006b; Sieber, enumerated the Japanese mycobiota of Discomycetes, a taxon no 2007), root endophytes are among the topics of a more recent focus longer used but still practical in the modern taxonomy, and enu- (Vandenkoornhuyse, Baldauf, Leyval, Straczek, & Young, 2002). merated 77 genera with 214 species in the order. Through our ex- Toju et al. (2013b) recognized the frequent occurrence of Helotiales ploration and enumeration, we added 57 newly documented taxa in plant roots based on a metagenomic analysis. Our study (Ho- in Japan, and 30 new taxa, including 2 genera (Otani, Hosoya, & soya, Hosaka, & Nam, 2017) on Fagus crenata, an endemic tree Furuya, 1991; Hosoya et al, 1993; Kubono & Hosoya, 1994; Hosoya species in Japan, also suggested the occurrence of Helotiales in F. & Otani 1995, 1997a, 1997b, 1997c; Hosoya & Harada 1999; Hosoya, crenata roots, while the majority of endophytes from leaves be- 2000, 2002, 2004, 2005, 2009; Ono & Hosoya, 2001; Tanaka & Ho- longed to Dothideales and Diaporthales. Currently, more research- soya, 2001; Hosoya & Huhtinen 2002; Narumi-Saito, Hosoya, Sano, ers are paying attention to Helotiales as root endophytes. The con- & Harada, 2006; Hosoya et al., 2010a, 2010c, 2011; Hosoya, Sung, nection between Phialocephala, a frequently occurring genus of Han, & Shin, 2010d; Han, Hosoya, & Shin, 2011; Hosoya et al., dark septate endophytes (DSE), and its mollisioid sexual state is 2012; Hosoya, Hosaka, Saito, Degawa, & Suzuki, 2013a; Hosoya et well known (Tanney, Bouglas, & Seifert, 2016). Rhizoscyphus ericae, al., 2013b; Hosoya, Saito, & Sasagawa, 2013c; Zhao, Hosoya, Shirou- which shows apparent symbiosis with plants, has recently been zu, Kakishima, & Yamaoka, 2013; Han et al., 2014a; Hosoya, Zhao, shown to be congeneric with (Fehrer, Réblová, Bam- & Degawa, 2014; Zhao & Hosoya, 2014, 2015; Degawa et al., 2015; basová, & Vohník, 2019; but see Baral & Kriegelsteiner, 2006). Gross, Hosoya, Zhao, & Baral, 2015; Hosoya & Zhao, 2016; Itagaki, Our preliminary studies showed that the majority of endophytes Nakamura, & Hosoya, 2019; Tochihara & Hosoya, 2019). These in- from F. crenata and Quercus crispula belonged to Dermateaceae s.l. cluded new species, new genera of both sexual and asexual states, and Hyaloscyphaceae s.l. (unpublished data). Thus, the above re- now being merged under single names (some examples are pre- ports seem reasonable. In fact, Bizabani and Dames (2015) showed sented in Fig. 2). that an isolate of Lachnum from Vaccinium roots, identified by molecular evidence, had plant growth promotion effects when 2.6. Monophyly of order and familial taxonomy re-inoculated. In most cases, identification of root endophytes is based on molecular evidence because most of the Helotiales lack an With the increasing molecular phylogenetic data for a wide asexual state and no morphologically distinguishing characteristic range of ascomycetes, it became possible to study large-scale phy- is available under culture. logeny at the order or higher level. After the exclusion of heteroge- Nakamura et al. (2018) detected a group in Hyaloscyphaceae neous elements, Helotiales became relatively homogeneous, but its (close to Hyphodicsus and ) that was dominant and monophyly was still not supported (Wang et al., 2006a; Wang, widely distributed in the roots of fagaceous trees, in addition to Johnston, Takamatsu, Spatafora, & Hibbett, 2006b) based on the Lachnum (Lachnaceae or Hyaloscyphaceae s.l.) and Rhizodermea combination of SSU, LSU, and 5.8S rDNA data. On the other hand, (Dermateaceae). In this study, I provided hitherto obtained se- Johnston et al. (2019), using up to 15 concatenated genes, con- quences of Helotiales used to determine environmental data to link firmed the monophyly of , the higher taxa that in- specimens, but no close match was found. Recognizing that the cludes Helotiales, but also demonstrated the paraphyly of the order. group obtained from the roots is phylogenetically distinguishable However, they also demonstrated a strong phylogenomic structure without any known reproductive morphology, Nakamura et al. within the order when genome information was used. Apparently, (2018) proposed several new species in Glutinomyces, originally

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A B C D

E EF G HI

J K JL M N

Figure 2. Representatives of Helotiales with taxonomic significance studied over the past 30 y in Japan. A–C: Gelatinipulvinella astraeicola. A monotypic genus with the yeast-like asexual state (B) with annellidic conidiation (C). It is a mycoparasite of Astraeus hygrometricus (Hosoya et al., 1995). D: longis- tipitata, a confused with Lachnum virgineum for a long time in Japan. It is currently regarded as an endemic species occurring in the cupules of Fagus crenata, an endemic tree. Although the fungus is strongly connected with the distribution of F. crenata (Hosoya et al., 2010b Gasca, Velez, & Hosoya, 2019), it does not show any population differentiation in contrast to its host. E: Venturiocistella japonica, occurring on Cercidiphyllum japonicum leaf. Its close phyloge- netic relationship with Glutinomyces, a root endophytic genus, is indicated (Nakamura et al., 2018). F–I: Hyphodicus hyaloscyphoides, a new species with a Cha- lara-like asexual state. are frequently detected in living plant roots. White apothecia on the substrate (F). Cross section of the apothecium, showing a typical structure of apothecium consisting of the stipe, excipulum, and hymenium composed of paraphyses and asci (G). Chalara-like conidiophores and co- nidia (H). Conidia are borne in chains or drops (I). J: , the pathogen of ash dieback. The photo was taken in a place where the first specimen of this fungus was collected (Sugadaira, Nagano Pref., Japan). Note that the blackened region of the host surface is the external view of the stroma. K: pseudosydowianum, originally recognized as an isolate of an unidentified endophyte, later connected with the specimens collected in the field, showing that barcoding data works (Hosoya et al., 2014). L: Lambertella corni-maris, the type of Lambertella, chiefly defined by having substratal stroma and spores that turn brown before germination. M: aurelia, the type species of its genus in the family Arachnopezizaceae. N: sp. This genus may contain a number of potentially undescribed species and are scarcely distinguishable in morphology because of their resemblances. Bars: B, C, G, H 10 μm; I 50 μm.

proposed by Nakamura (2017) for a helotialean fungus for which dicus. For the group including these genera, Hyphodiscaceae was only the hyphal state was known. Later, Nakamura et al. (2019) proposed (Ekanayaka et al. 2019). demonstrated that Glutinomyces exchanges its genetic material through a parasexual process. These findings suggested a lack of 3.3. Ash dieback information to link the members occurring as root endophytes, but they also suggested that there are groups of Helotiales that have Ash dieback is an emerging plant tree disease with severe mor- adapted to underground life, losing morphologically differentiated tality for European or common ash trees ( excelsior). Be- sexual/asexual reproductive structures. Another good example is cause F. excelsior is one of the key components of forest ecosystems Meliniomyces, described based on sterile morphotypes with charac- and also economically important for timber production, the spread teristic pinwheel-like colonies. These are common root endophytes of ash dieback is very concerning. Nevertheless, the disease started and were recently combined with Hyaloscypha (Fehrer et al., 2019). to spread from Poland in 1992, and now covers almost the complete Fehrer et al. (2019) also reported the first observation of an asexual distribution range of Fraxinus in Europe (for a recent review, see state for Hyaloscypha bicolor, induced in vitro after incubation at 6 Enderle, Stenlid, & Vasaitis, 2019). °C. The pathogen is now known as Hymenoscyphus fraxineus (T. It is also worthwhile to mention that Hyalopeziza in Nakamura Kowalski) Baral, Queloz & Hosoya (Baral, Queloz, & Hosoya, et al. (2018) was represented by Hyalopeziza pygmaea previously 2014), but it has a confusing nomenclatural history. Originally it transferred to propose a new genus Hyphopeziza (Han et al. 2014a) was described as an asexual fungus, Chalara fraxineus T. Kowalski showing an intermediate feature between Hyalopeziza and Hypho- (Kowalski, 2006), once connected with

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(Gillet) W. Phillips (Kowalski & Holdenrieder, 2009) in its sexual numerous new natural compounds (Bycroft & Payne, 2013). state. Later, Queloz et al. (2011) discovered a cryptic entity within Regarding discovery research, the majority of the results have H. albidus corresponding to C. fraxineus and proposed a new spe- come from soil fungi, in particular Aspergillus and Penicillium, and cies H. pseudoalbidus Queloz, Grünig, Berndt, T. Kowalski, T.N. little research has focused on Helotiales. Although current environ- Sieber & Holdenr. distinguished from H. albidus on a molecular mental DNA studies suggest the occurrence of Helotiales in the basis. Baral (pers. comm.) kindly informed me of the possibility of soil, they cannot be isolated from soils by conventional techniques, the conspecificity of H. pseudoalbidus and Lambertella albida [one whereas obtaining isolates from ascospores is relatively easy. Helo- of the synonyms of H. albidus, documented as a new record from tiales are therefore a good example of an “underutilized, but easily Japan (Hosoya et al., 1993)]. As a result of our genetic comparative accessible fungi,” as proposed by Bergstom et al. (1995). Another studies, the conspecificity of L. albida documented in Japan and H. expectation for Helotiales is that they may produce metabolites pseudoalbidus was demonstrated (Zhao, Hosoya, Baral, Hosaka, & with different structures from those of Aspergillus and Penicillium Kakishima, 2012). Later, because of the change in nomenclature because they significantly differ in their phylogenetic position. following the One Fungus One Name (1F = 1N) policy (Hawk- Stadler and Anke (1993, 1995) were among the pioneers in uti- sworth, 2011; Norvell, 2011), the new combination, H. fraxineus, lizing Helotiales and discovered a series of chlorinated metabolites. was proposed (Baral et al., 2014). Our attempts succeeded in the discovery of various enzyme inhibi- Zhao et al. (2012) also demonstrated greater genetic diversity in tors, such as squalene synthase (Hosoya et al., 1997; Tanimoto et the Japanese population compared to that of the European popula- al., 1997), sphingomyelinase (Tanaka, Nara, Suzuki-Kongai, Ho- tion. This supported the possible introduction of H. fraxineus into soya, & Ogita, 1997; Nara, Tanaka, Hosoya, Suzuki-Konagai, & Og- Europe from Asia, including Japan, which was suggested by Hus- ita, 1999), sphigosine kinase (Kono, Tanaka, Ogita, Hosoya, & Ko- son et al. (2011), Queloz et al. (2011), and Timmermann et al. hama, 2000; Kono et al., 2001), testosterone reductase (Hosoya et (2011). Our discovery of H. fraxineus in Japan long before the emer- al., 1999), antibacterials (Matsumoto, Hosoya, & Shigemori, 2010; gence of the fungus in Europe partially supported this hypothesis. Matsumoto, Hosoya, Tomoda, Shiro, & Shigemori, 2011; Tanabe et Later, the distribution of H. fraxineus in Asia became even wider al., 2015; Kawashima, Hosoya, Tomoda, Kita, & Shigemori, 2018), after being found in Korea (Han et al., 2014b) and China (Zheng & plant growth regulation factors (Tanabe, Matsumoto, Hosoya, Sato, Zhuang, 2014). Furthermore, Gross et al. (2014) conducted a de- & Shigemori, 2013), and antifungals (Ohyama et al., 2002). tailed population genetic analysis to compare European and Japa- Concerning ash dieback, several metabolites from H. fraxineus, nese populations, and postulated that the establishment of the Eu- including cytotoxic compounds and mycotoxins have been discov- ropean population was based on the transfer of a small population ered [see Surup et al. (2018) for hyfraxins (cytotoxic) and other through the “founder effect.” This was later confirmed by genomic compounds]. analysis by McMullan et al. (2018), who postulated that the initial With the advent of high-throughput screening coupled with population was founded by only two individuals. Their studies also natural product libraries, the research interest in search of new suggested the presence of more than 1000 effectors that dispute the natural products has declined (Harvey, 2008). However, there is the resistance of the plant host. Together with the discovery research of expectation that natural products are not lost with the application bioactive metabolites (see Surup et al. 2018 and other papers cited of combinatorial chemistry, genetic engineering, and drug design herein), the process for symptom development has been elucidat- technology (Pawar, 2014). ed. In addition, multiple species of Lachum have been shown to Furthermore, the possibility of the endophytic occurrence of produce pigments and polysaccharides that exhibit antioxidant and the fungus was examined, because no symptoms have been ob- antimicrobial activities (Ming, Wei, Cong, Shi-yan, & Yang, 2009; served in Fraxinus trees where the first occurrence of H. fraxineus Ye, Peng, Fang, Li, & Yang, 2009; Qiu, Ma, Ye, Yuan, & Wu, 2013) in Japan was reported. With a clear experiment in combination providing remarkable support for Helotiales as a good biological with field observations, Inoue et al. (2018) confirmed the occur- resource. rence of H. fraxineus as an endophyte in living leaves of F. mand- shurica in Japan. 5. Future perspectives A combination of the characteristics of H. fraxineus, mainly having substratal stroma and ascospores turning brown after being As a result of exploration, collected specimens, whether they are discharged before germination, apparently satisfied the disposition identified or unidentified, have cumulated in the museum fun- of this fungus as Lambertella (Zhao et al., 2012). However, as garia. The occurrence data, based on specimens or observations, shown by Zhao et al. (2016), these characters were the consequence are being accumulated in public databases (e.g., GBIF, https:// of convergent evolution, and the combination of these characters www.gbif.org/), and can be utilized for various purposes, including no longer supports the disposition of this fungus to Lambertella. taxonomic and ecological research. Cumulation of isolates and/or Since Hymenoscyphus is a large genus it may be divided into multi- parts of specimens and genetic data are also compiled in separate ple genera in the future. databases (see 2.1). However, once digitized, interoperability be- tween the data increases, and more integrated research is possible. 4. Discovery Research Fungi occur in various environments, including the Helotiales. Metagenomic research has revealed the potential occurrence of Because fungi produce a great number of metabolites, they have Helotiales in various environments where we have seldom suspect- been a significant resource in natural product research, and a num- ed them (Peay, 2014) and provide an opportunity to uncover eco- ber of compounds with novel structures and/or activities have been logical functions (Almario et al., 2017). Rapidly increasing data discovered (Dreyfuss & Chapela, 1994). Regarding the motivation generated by next-generation sequencing provides insights into the of recent research, three major interests are recognized: natural interactions between fungi and plants (Toju et al., 2013a, 2013b). product chemistry, chemicals from intermediate plant-fungal inter- Therefore, when the collected specimens are accompanied by ge- actions, and discovery research for compounds for drugs (Hosoya, netic data, such as barcoding sequences, the specimens could be 1998). These research interests overlap and lead to discoveries of exploited as vouchers to provide biological data. We have estab-

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lished links between specimen occurrence, morphological infor- Baral, H. O., & Krieglsteiner, G. J. (1985). Bausteine zu einer Askomyzeten-Flora der mation, and DNA barcoding data (Hosoya, Jinbo, & Tanney, 2015). Bundesrepublik Deutschland: in Suddeutchland gefundene inoperculate Dis- komyceten mit taxonomischen, oekologischen und chorologischen Hinweisen. In the near future, it will be more important to exploit the distrib- Beiheft zur Zeitschrift für Mykologie, 6, 1–226. uted data in a more integrated manner, using DNA barcoding se- Baral, H. O., & Krieglsteiner, L. (2006). Hymenoscyphus subcarneus, a little known quences as possible clues for linkages. In fact, we succeeded in bryicolous discomycete found in the Bialowieza National Park. Acta Mycologi- linking unnamed endophytic fungi with newly described species ca, 41, 11–20. (Hosoya et al., 2014). Baral, H. O., Queloz, V., & Hosoya, T. (2014). Hymenoscyphus fraxineus, the correct Although some shortcomings are indicated by Hofstetter et al. scientific name for the fungus causing ash dieback in Europe. IMA Fungus, 5, 79-80. (2019), barcoding for fungi using ITS-5.8S sequences has been Bergstom, J. D., Dufresne, C., Bills, G. F., Nallin-Omstead, M. & Byne, K. (1995). widely accepted (Schoch et al., 2012), and there are ongoing initia- Discovery, biosynthesis, and mechanism of action of the zaragozic acids: potent tives to link the DNA sequence data with the data from specimens inhibitors of squalene synthase. Annual Review of Microbiology, 49, 607–639. (e.g., UNITE, Kõljalg et al., 2005). However, whether all fungal en- Bizabani, C., & Dames, J. A. (2015). Effects of inoculating Lachnum and tities should have Linnean binominals is a matter of debate (Mon- isolates on the growth of Vaccinium corymbosum. Microbiological Research, ey, 2013). Fungal biodiversity is vast, and it is apparent that mil- 181, 68–74. Bycroft, B. W., & Payne, D. J. (2013). Dictionary of antibiotics and related substances lions of species could be recognized. It is therefore of substantial with CD-ROM (2nd ed.). Oxford: Taylor and Francis. value to provide a common identifier, such as a digital objective Cantrell, S. A., & Hanlin, R. T. (1997). Phylogenetic relationships in the family Hy- identifier (DOI), as has been done in UNITE (Kõljalg et al., 2005) to aloscyphaceae inferred from sequences of ITS regions, 5.8S ribosomal DNA the recognized groups of possible species (species hypothesis) to and morphological characters. Mycologia, 89, 745–755. recognize the biological group as an entity. However, more speci- Degawa, Y., Hosoya, T., Hosaka, K., Hirayama, Y., Saito, Y., & Zhao, Y. J. (2015). Re- mens must be collected to ensure the actual existence of groups. As discovery of Roesleria subterranea from Japan with a discussion of its infraspe- cific relationships detected using molecular analysis. Mycokeys, 9, 1–9. Korf (2005) encouraged the mycologist, we must “collect, collect, Dreyfuss, M. M., & Chapela, I. H. (1994). Potential of fungi in the discovery of novel, and collect” information to understand the biodiversity of Helotia- low-molecular weight pharmaceuticals. Biotechnology, 26, 49–80. les. After all, it seems for a discosystematist, Japan is still a “rela- Dumont, K. P. (1971). Sclerotiniaceae II. Lambertella. Memoirs of the New York Bo- tively unexplored paradise” (Korf, 1958). tanical Garden, 22, 1–178. Ekanayaka, A. H., Hyde, K. D., Gentekaki, E., McKenzie, E. H. C., Zhao, Q., Bulga- kov, T. S., et al. (2019). Preliminary classification of Leotiomycetes. Mycosphere, Disclosure 10, 310–489. Enderle, R., Stenlid, J., & Vasaitis, R. (2019). An overview of ash (Fraxinus spp.) and The authors declare no conflicts of interest. All the experiments the ash dieback disease in Europe. CAB Reviews, 14, 1–12. undertaken in this study complied with the current laws of the Eriksson, O. E., Baral, H. O., Currah, R. S., Hansen, K., & Kurtzman, C. P. (2003). county where they were performed. Notes on ascomycete systematics. Nos 3580–3623. Myconet, 9, 1–23. Fehrer, J., Réblová, M., Bambasová, V., & Vohník, M. (2019). The root-symbiotic Rhizoscyphus ericae aggregate and Hyaloscypha (Leotiomycetes) are congener- Acknowledgments ic: Phylogenetic and experimental evidence. Studies in , 92, 195–225. Gasca, J., Velez, P., & Hosoya, T. (2019). Phylogeography of post-Pleistocene popula- The present article is based on the memorial talk for the recep- tion expansion in Dasyscyphella longistipitata, an endemic fungal symbiont of tion of MSJ award. I express my deepest gratitude to the late Keis- Fagus crenata in Japan. Mycokeys, 65, 1–24. uke Tubaki, Prof. Emeritus, the University of Tsukuba, who intro- Gross, A., Hosoya, T., & Queloz, V. (2014). Population structure of the invasive forest duced me to the world of fungi, and members of Mycology pathogen Hymenoscyphus pseudoalbidus. Molecular Ecology, 23, 2943–2960. Gross, A., Hosoya, T., Zhao, Y. J., & Baral, H. O. (2015). Hymenoscyphus linearis sp. Laboratory, University of Tsukuba for my fundamental education. nov.: another close relative of the ash dieback pathogen H. fraxineus. Mycologi- I also express my sincere thanks to Prof. Seiji Tokumasu, University cal Progress, 14, 1–15. of Tsukuba, who provided opportunities for discussion of fungal Han, J. G., Hosoya, T., & Shin, H. D. (2011). castaneae sp. nov. (Hyalos- ecology and many insights into the behavior of fungi. I am also in- cyphaceae, Helotiales) on Japanese chestnut bur. Mycotaxon, 118, 89–94. debted to Dr. Yoshio Otani, Researcher Emeritus of the National Han, J. G., Hosoya, T., Sung, G. H., & Shin, H. D. (2014a). Phylogenetic reassessment Museum of Nature and Science for his guidance and teaching me of Hyaloscyphaceae sensu lato (Helotiales, Leotiomycetes) based on multigene analyses. Fungal Biology, 118, 150–167. the taxonomic background of Helotiales. Dr. Kouhei Furuya, the Han, J. G., Shrestha, B., Hosoya, T., Lee, K. H., Sung, G. H, & Shin, H. D. (2014b). former Director of Tsukuba Research Laboratories in Sankyo Co. is First report of the ash dieback pathogen Hymenoscyphus fraxineus in Korea. greatly appreciated for his kind advice and guidance in research. Mycobiology, 42, 391–396. Finally, I express my gratitude to the members of the Plant Pathol- Harada, Y. (1977) Studies on the Japanese species of (Sclerotiniaceae). ogy Laboratory, University of Tsukuba, mycology group of Sankyo Bulletin of the Faculty of Agriculture, Hirosaki University, 27, 30–109. Co., Ltd., and members of Department of Botany in the National Harvey, A. L. (2008). Natural products in drug discovery. 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